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UV-Vis absorption changes when the cuvette direction changes
Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.
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When working with UV-Vis photo-spectrometer instruments, I have heard that the cuvette must be positioned only one direction in the chamber. I try to turn its direction, sometimes the signal intensity is changed, about 0.5% or so, and sometimes not. How can you explain this phenomenon?
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I'd go further; it's best not to move the cuvette at all if you're being really fussy.
My guess:
If the light beam hits the cuvette exactly at right-angles and the cuvette has perfectly flat walls, inside and out, then there should be no refraction and no deviation of the light-beam. Any slight angling of the cuvette will displace the beam slightly sideways, which means a different amount of the total light will get selected by any slits downstream of the cuvette. (I'm assuming there's no reflection on any surface; that's another issue). So any displacement of the cuvette is a problem because it could, potentially, change the displacement of the light beam. If the cuvette had perfect rotational symmetry (ideally perfectly square) and could be placed perfectly, it wouldn't matter which way round it went, but nothing is perfect, and it's quite possible there are very slight angles and curves on the windows, and that they will differ very very slightly.
(Also, for that matter, if you imagine that the beam is probably not perfectly parallel through the cuvette, if the two sides of the cuvette have slightly different absorbances, it would matter which came first.. I rather doubt this is the source of the problem though)
BUT, if you want to keep the cuvette without moving it, how do you intend to wash it and dry it between solutions? It's easy to create a 0.5% error by failing to ensure that 100% of the liquid in the cuvette is the stuff you are trying to measure, and not a washing solvent or the last sample...
My guess:
If the light beam hits the cuvette exactly at right-angles and the cuvette has perfectly flat walls, inside and out, then there should be no refraction and no deviation of the light-beam. Any slight angling of the cuvette will displace the beam slightly sideways, which means a different amount of the total light will get selected by any slits downstream of the cuvette. (I'm assuming there's no reflection on any surface; that's another issue). So any displacement of the cuvette is a problem because it could, potentially, change the displacement of the light beam. If the cuvette had perfect rotational symmetry (ideally perfectly square) and could be placed perfectly, it wouldn't matter which way round it went, but nothing is perfect, and it's quite possible there are very slight angles and curves on the windows, and that they will differ very very slightly.
(Also, for that matter, if you imagine that the beam is probably not perfectly parallel through the cuvette, if the two sides of the cuvette have slightly different absorbances, it would matter which came first.. I rather doubt this is the source of the problem though)
BUT, if you want to keep the cuvette without moving it, how do you intend to wash it and dry it between solutions? It's easy to create a 0.5% error by failing to ensure that 100% of the liquid in the cuvette is the stuff you are trying to measure, and not a washing solvent or the last sample...
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